ABSTRACT Cryptosporidium parvum and C. hominis are common causative agents of chronic diarrheal disease in immunocompromised patients such as those with HIV/AIDS. Although C. parvum is primarily acquired from animals, infection with C. hominis occurs exclusively in humans. Unfortunately, the only FDA-approved drug for cryptosporidiosis is not effective in controlling infection in immunocompromised patients. Progress in studying the biology of C. hominis is hampered by the lack of small animal models or in vitro systems for long- term cultivation. Limited replication of C. hominis does occur in adenocarcinoma lines; however, these tumor cell lines do not mimic the normal physiology, metabolism, or cellular differentiation of human epithelium. The normal replicative niche for C. parvum and C. hominis is the intestinal epithelial cell, and infection is primarily concentrated in the small intestine. In preliminary studies we have shown that stem cell-derived cultures of primary mouse Intestinal Epithelial Cells (IECs) allow complete development and long-term culture of C. parvum in vitro. C. parvum undergoes both asexual and sexual phases of development in mouse IEC cultures, culminating in production of oocysts. Unfortunately, these mouse IECs do not support the growth of C. hominis, presumably due to the narrow host range of this parasite. The proposed project will adopt these advances with C. parvum to develop a similar cultivation system for C. hominis using human-derived IECs. We will also profile development of C. hominis using a bank of existing monoclonal antibodies and stage- specific gene expression markers to define specific developmental stages. These markers will provide qualitative and quantitative indicators for establishing conditions for in vitro propagation of C. hominis. If successful, these studies have the potential to be transformative in providing the first in vitro model for cultivation of C. hominis.